The N-Terminal GYPSY Motif Is Required for Pilin-Specific Sortase SrtC1 Functionality in Lactobacillus rhamnosus Strain GG

Abstract

Predominantly identified in pathogenic Gram-positive bacteria, sortase-dependent pili are also found in commensal species, such as the probiotic-marketed strain Lactobacillus rhamnosus strain GG. Pili are typically associated with host colonization, immune signalling and biofilm formation. Comparative analysis of the N-terminal domains of pilin-specific sortases from various piliated Gram-positive bacteria identified a conserved motif, called GYPSY, within the signal sequence. We investigated the function and role of the GYPSY residues by directed mutagenesis in homologous (rod-shaped) and heterologous (coccoid-shaped) expression systems for pilus formation. Substitutions of some of the GYPSY residues, and more specifically the proline residue, were found to have a direct impact on the degree of piliation of Lb. rhamnosus GG. The present findings uncover a new signalling element involved in the functionality of pilin-specific sortases controlling the pilus biogenesis of Lb. rhamnosus GG and related piliated Gram-positive species.

Fig 1. Sequence alignment of the N-terminal domains of pilin-specific sortases from various piliated Gram-positive species and LOGO motif analysis.

(A) The alignment was generated using MUSCLE [41]. The so-called GYPSY motif is highlighted in yellow, with the tyrosine residue and the proline residue constituting the functional core of the motif. Residues with a partial degree of conservation within the GYPSY motif are shaded in light grey. The start methionine is highlighted in green. Protein sequences were obtained from public databases, as indicated in the text. In the table, the start of the three protein sequences marked with an asterisk (*) were re-analyzed in the present study. In addition, all sortase sequences excluding E. faecium SrtC4, S. pneumoniae TIGR4 SrtD and S. agalactiae 2603V/R sag0650 were analyzed using the MEME suite [42], resulting in the LOGO motif depicted in (B). Legend: blue, hydrophobic residue; green, polar, non-charged residue; magenta, acidic residue and red, positively charged residue.

Fig 2. Modelling of the region containing the GYPSY motif of the Lb. rhamnosus GG SrtC1 with kinked proline residue.

(A) Output prediction results using PSIPRED server [43,44] indicated that the GYPSY motif introduces a breakage within an α-helix as opposed to some mutant variants; (B) and (C), ab initio model of the GYPSY motif. The kinked proline residue is shaded in fuchsia. The relevant AA modification(s) introduced in the present study are shown in vivid green.

Fig 3. Electron microscopy observations of L. lactis NZ9000 producing various SpaA pilus structures.

Bacterial cells were immunogold-labeled with anti-SpaA serum and gold particles (10 nm). Legend: A, NZ9000 + SpaA-SrtC1; B, NZ9000 + SpaA-SrtC1^{Y29G, P30G}; C, NZ9000 + SpaA-SrtC1^P30G; D, NZ9000 + SpaA-SrtC1^Y29G.

Fig 4. Western blotting analysis of L. lactis NZ9000 expressing different spaA-srtC1 gene variants, where the GYPSY motif has been altered by AA substitution.

SpaA pilin proteins were detected using anti-SpaA polyclonal antibodies. Legend: #, protein weight marker; HMWL, high molecular weight ladder; *, band corresponding to elongated pilus structures.

Fig 5. Electron microscopy observations of Lb. rhamnosus PB12 and its SrtC1-complemented derivatives.

Bacterial cells were immunogold-labeled with anti-SpaA serum and gold particles (10 nm). Legend: A, Lb. rhamnosus GG; B, Lb. rhamnosus PB12; C, Lb. rhamnosus PB12 + SrtC1.

Fig 6. Electron microscopy observations of Lb. rhamnosus PB12 complemented with SrtC1 variants.

Bacterial cells were immunogold-labeled with anti-SpaA serum and gold particles (10 nm). Legend: A, Lb. rhamnosus PB12 complemented with SrtC1 ^{Y29G, P30G}; B, Lb. rhamnosus PB12 complemented SrtC1 ^Y29G; C, L. rhamnosus PB12 complemented SrtC1 ^P30G.

Fig 7. Western blotting analysis of Lb. rhamnosus GG and PB12 expressing different srtC1 gene variants, where the GYPSY motif has been altered by AA substitution.

SpaA pilin proteins were detected using anti-SpaA polyclonal antibodies. Legend: #, protein weight marker; HMWL, high molecular weight ladder; *, band corresponding to elongated pilus structures.

Fig 8. Mucus binding assays of Lb. rhamnosus GG and PB12 expressing different srtC1 gene variants, where the GYPSY motif has been altered by AA substitution.

The averages and standard deviations were obtained from a total of twelve technical replicates from three biological replicates.

Fig 9. Western blotting analysis of L. lactis MG1363 PPiA KO mutant and its complemented mutant, where the SpaA-SrtC1 cassette was introduced.

SpaA pilin proteins were detected using anti-SpaA polyclonal antibodies. Legend: #, protein weight marker; HMWL, high molecular weight ladder; *, band corresponding to elongated pilus structures.